Proteins and peptides make up a large part of the armamentarium available for the molecular imaging of cell-surface biomarkers. Targeted proteins produced by genetic engineering are very attractive as PET imaging agents, but labeling with conventional 18F-based prosthetic groups is problematic due to long synthesis times, poor radiochemical yields, and low specific activity. Although the development of “ideal” imaging agents is an important goal, in practice many imaging agents are developed from existing proteins, such as monoclonal antibodies (Mabs) and their engineered fragments, which are initially developed as potential therapeutic agents or to explore a target's biology. PET imaging agents may function in diagnostic assays or biomarker tests, to enable patient selection, inform decisions around indication choice for therapeutic candidates, and maximize clinical benefit of a therapeutic agent that targets the same receptor or disease pathway. Predictive biomarker tests are conducted before treatment to predict whether a particular treatment is likely to be beneficial. Prognostic biomarkers are correlated with disease outcome and may improve clinical trial design and treatment, and data interpretation confidence levels.
The development of Positron Emission Tomographic (PET) imaging agents from a Mab template (Immuno-PET) holds promise as a tool for localizing and quantifying molecular targets and may enhance the non-invasive clinical diagnosis of pathological conditions (van Dongen et al (2007) Oncologist 12; 1379-89; Williams et a (2001) Cancer Biother Radiopharm 16:25-35; Holliger et al (2005) Nat Biotechnol 23:1126-36). PET is a molecular imaging technology that is increasingly used for detection of disease. PET imaging systems create images based on the distribution of positron-emitting isotopes in the tissue of a patient. The isotopes are typically administered to a patient by injection of probe molecules that comprise a positron-emitting isotope, such as F-18, C-11, N-13, or O-15, covalently attached to a molecule that is readily metabolized or localized in the body (e.g., glucose) or that chemically binds to receptor sites within the body. In some cases, the isotope is administered to the patient as an ionic solution or by inhalation. Small immuno-PET imaging agents, such as Fab antibody fragments (50 kDa) or diabodies, paired dimers of the covalently associated VH-VL region of Mab, 55 kDa (Shively et al (2007) J Nucl Med 48:170-2), may be particularly useful since they exhibit a short circulation half-life, high tissue permeability, and reach an optimal tumor to background ratio between two to four hours after injection facilitating the use of short half-life isotopes such as the widely available 18F (109.8 min).
Targeted proteins produced by genetic engineering are very attractive as PET imaging agents, but labeling with conventional 18F-based prosthetic groups is problematic due to long synthesis times, poor radiochemical yields, and low specific activity. a modular platform for rapid preparation of various water-soluble prosthetic groups capable to efficiently introduce 18F into proteins. Combining the sensitivity and high-resolution offered by 18F-based PET imaging with the high specificity of these antibody fragments is a particularly attractive strategy for the research and clinical development of novel diagnositic assays and therapeutics. Production of 18F-labeled proteins by current methods is inadequate because relatively mild aqueous reaction conditions are necessary to preserve the function of most proteins. Existing 18F-labeled prosthetic groups used for protein conjugations are often limited by some combination of poor radiochemical yield, long synthesis time, and low specific activity. Improved methods for generating 18F-labeled proteins are valuable in facilitating molecular imaging in humans to address clinical development processes such as level of target expression, heterogeneity and course of expression.
Site-specific conjugation is preferred over random amino modification as it enables chemical modification of a site away from the binding site, promoting complete retention of biological activity and allowing control over the possible number of prosthetic groups added. Genetically engineered proteins containing cysteine at select positions have been investigated for developing site-specific conjugations (Junutula, J. R. et al (2008) J Immunol Methods 332:41-52; US 2007/0092940). The radionuclide labelling of thiol groups on engineered cysteine residues by prosthetic groups is advantageous as a site-specific method since the presence of cysteine is limited within the proteome, of which the non-reactive disulfide form is dominant (Olafsen et al (2004) Protein Eng Des Sel 17:21-7; Tait et al (2006) J Nucl Med 47:1546-53; Li et al (2008) Bioconjug Chem 19:1684-8). A protein engineering method, PHESELECTOR, employs phage display library to select the optimal amino acid position for cysteine substitution (Junutula et al. (2008) Nat Biotechnol 26:925-32; Junutula et al (2008) J Immunol Methods 332:41-52; US 2007/0092940). Using the PHESELECTOR method, protein stability and binding affinity is retained while the formation of undesired disulfide bonds is minimized providing optimal conjugation efficiencies. This method is used to produce modified Mab containing an available cysteine (ThioMab), from which a Fab fragment (ThioFab) with a reactive thiol group is conveniently generated.
Prosthetic groups containing the maleimide group, such as [18F]FBEM (Cai et al (2006) J Nucl Med 47:1172-80), [18F]FBAM (Berndt et al (2007) Nucl Med Biol 34:5-15), [18F]FBABM (Li et al (2008) Bioconjug Chem 19:1684-8; Toyokuni et al (2003) Bioconjug Chem 14:1253-9), [18F]FBOM (Wuest et al (2009) Amino Acids 36:283-295), [18F]FDG-MHO (Wuest et al (2008) Bioconjug Chem 19:1202-10), and [18F]FPyMe (de Bruin et al (2005) Bioconjug Chem 16:406-20), have been used to site-specifically introduce 18F into a thiol-bearing protein. These labelling reagents [18F]FBEM, [18F]FBAM, [18F]FBABM, and [18F]FBOM were developed from a common platform where the aromatic precursor, F-fluorobenzaldehyde ([18F]FBALD), is coupled with an aminooxy-bearing maleimide precursor. However, the presence of aromatic and (with the exception of [18F]FBOM) aliphatic moieties enhances the lipophilicity of these prosthetic groups, potentially limiting the conjugation efficiency with protein thiol groups present within a hydrophilic environment. Furthermore, these prosthetic groups require long synthesis times and typically provide relatively poor radiochemical yields of 18F-labeled protein.